The overall goal of this research program is to understand the role of K+ channels in the regulation of insulin secretion. Excitation-secretion coupling in insulin-secreting beta-cells is a calcium-dependent process that involves a dynamic interplay between K+ and Ca 2+ channels. Ca 2+ channels mediate the action potential and K+ channels regulate the membrane potential (Vm). Following glucose stimulation the beta-cell depolarizes, activating voltage-dependent Ca 2+ and K+ channels, beta-cells then display characteristic oscillations of electrical activity consisting of depolarized plateaus upon which are superimposed bursts of Ca 2+ channel-mediated action potentials, interrupted by periodic intervals of K+ channel-mediated repolarization. Repolarization of Vm is critical in the regulation of insulin secretion, but knowledge of the specific K+ channels involved is incomplete, particularly in human islets. We have shown that the Kv2.x family (Kv2.1 and Kv2.2) of delayed rectifier voltage-dependent K+ (Kv) channels plays an essential role in beta-cell repolarization and insulin secretion. We propose to test the role of specific Kv2 isoforms and their modulators in beta-cell physiology. The specific aims are divided into two areas: further studies in mouse islets, and studies in human islets. (Aim 1) To test the hypothesis that Kv2.x channels regulate the Kv current of mouse beta-cells. Kv2.x channels will be specifically inhibited by expression of dominant-negative mutant Kv2.1 and Kv2.2 cDNAs in mouse islet cells in vitro, and the effects on Vm intracellular concentration, and insulin secretion will be determined. Kv2.x expression will be inhibited in transgenic mouse models using beta-cell specific promoters, and islet function assessed in vitro and in vivo. (Aim 2) To define the role of Kv channels in human beta-cell function. Kv channels and accessory subunits expressed in fluorescently-sorted human beta-cells will be identified. Their roles in beta-cell signaling will be characterized pharmacologically and further characterized by targeting expression of dominant-negative mutant Kv constructs using viral transduction. We anticipate that our studies will provide a better understanding of the underlying molecular mechanisms that regulate insulin secretion and will facilitate development of new therapeutic strategies to treat diabetes.